Estrogen is a crucial hormone in the human body, regulating many diverse physiological effects, including the growth, development and homeostasis of numerous tissues. Estrogen plays a critical role in the development of normal breast tissue, whereas responsiveness to estrogen represents one of the most important prognostic and therapeutic factors for patients with breast cancer. Many of these properties have been attributed to a soluble receptor for estrogen (ER) that functions in large part as a ligand-activated transcription factor. We have characterized a novel intracellular, 7-transmembrane spanning, G protein- coupled estrogen receptor (GPR30) that we propose functions alongside ER to regulate cell and tissue responsiveness to estrogen. We have discovered that GPR30 resides predominantly in the endoplasmic reticulum. The goal of Aim 1 is to determine the sequences within GPR30 that regulate its cellular location. We have also developed a novel family of fluorescent estrogen derivatives that binds to both the ER and GPR30. In Aim 2 we will use these fluorescent estrogens to develop cell-based estrogen binding assays for GPR30 and determine the binding specificities and affinities of a collection of estrogen-related compounds. Recently, we have characterized novel GPRSO-mediated signal transduction pathways in breast cancer cells. The nuclear accumulation of phosphatidylinositol 3,4,5-trisphosphate is initiated by estrogen-mediated transactivation of epidermal growth factor receptor (EGFR) by GPR30. The goal of Aim 3 is to investigate the mechanisms involved in EGFR transactivation with respect to EGFR phosphorylation and internalization, leading to transcriptional activation and cell growth control. Finally, we have established a colony of GPR30 null mice to dissect the in vivo function of GPR30 in breast growth and development. In Aim 4, we will characterize GPR30 expression in wild type mice and analyze the phenotypes of GPR30 null mice. We will also characterize the phenotypes of mice in which both GPR30 and the classical ERs have been deleted. With these tools, we can address estrogen-responsive signaling of GPR30 in a genetically defined background. The experiments proposed will be invaluable in defining the roles of this novel estrogen- responsive receptor in estrogen-mediated growth, proliferation, and differentiation in normal development as well as in neoplastic progression and the treatment of breast cancer.